| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* School of Clinical Sciences, University of Liverpool, Liverpool, UK;
Department of Medicine, University of California, San Diego, California, USA; and
Department of Physiology, Loyola University, Chicago, Illinois, USA
1 Correspondence: Division of Metabolic and Cellular Medicine, School of Clinical Sciences, University of Liverpool, Liverpool L69 3GA, UK. E-mail: mdcr02{at}liv.ac.uk
SPECIFIC AIMS
Skeletal muscle aging is characterized by atrophy, a deficit in specific force generation, increased susceptibility to injury, and incomplete recovery after severe injury. It has been proposed that the accumulation of oxidation products in muscle and the inability to activate redox-sensitive transcription factors play a major role in age-related muscle dysfunction. Transgenic overexpression of heat shock protein (HSP)70 throughout life protects against the age-related fall in maximum force and facilitates successful recovery in comparison with muscles of age-matched wild-type (WT) mice. The aim of this study was to examine muscles of quiescent adult and old WT and HSP70 overexpressor mice for markers of oxidative damage and to examine the ability of these muscles to activate nuclear factor-
B in response to nondamaging contractile activity.
PRINCIPAL FINDINGS
1. Markers of oxidative damage were elevated in anterior tibialis muscles of old WT mice compared with muscles of adult WT mice
The study used adult (10–12 mo) and old (26–28 mo) male and female WT B6XSJL and transgenic mice that had a chimeric transgene that consisted of an inducible HSP70 gene of a rat under a ßbeta;-actin promoter. This resulted in a 10- to 20-fold increase in HSP70 content of anterior tibialis (AT) muscles in adult and old HSP70 overexpressors compared with that of age-matched WT mice. Catalase and total superoxide dismutase (SOD) activity and muscle glutathione (glutathione) and protein thiol content of muscles were measured spectrophotometrically. Protein carbonyl content was determined by electrophoresis and Western blotting. Data demonstrated that the total SOD and catalase activity was significantly increased in quiescent muscles from old WT mice (Fig. 1
). The total glutathione content of quiescent muscles of old WT mice was significantly lower than that of adult WT mice (Fig. 1)
, and the malonaldehyde and protein carbonyl content of muscles of WT mice increased significantly with age (Fig. 1)
.
|
2. Age-related changes in markers of oxidation in muscles of old WT mice were not evident in muscles of old HSP70 overexpressor mice
No effect of age was seen in SOD and catalase activity, glutathione malonaldehyde, or carbonyl content of muscles of HSP70 overexpressor mice (Fig. 1)
.
3. A nondamaging but demanding isometric contraction protocol resulted in increased NF-kB DNA binding in muscles of adult WT mice, but this increase was abolished in muscles of old WT mice
Muscles of anesthetized mice were electrically stimulated to contract for 15 min with square wave pulses of 0.1 millisecond duration at 100 Hz and 70 V for 0.5 s every 5 s. Mice were then killed, and muscles were removed. DNA binding activity of NF-B was determined in nuclear extracts of muscles using EMSA. The DNA binding activity for NF-B was elevated after contractile activity in muscles of the adult WT mice (Fig. 2
). In contrast, muscles of old WT mice showed no change in NF-B binding activity in response to the isometric contraction protocol (Fig. 2
).
|
4. Overexpression of HSP70 in muscles resulted in a preservation of the activation of NF-kB binding in muscles of old HSP70 overexpressor mice
NF-B DNA binding activity of nuclear extracts from old HSP70 overexpressor mice was significantly elevated after contractile activity to a comparable concentration as adult mice (Fig. 2)
.
CONCLUSIONS AND SIGNIFICANCE
These data are novel and provide a fundamental insight into the mechanisms responsible for the well-documented age-related failure in muscle function and recovery from damage. This study has demonstrated that the dramatic improvement in the functional capacity of muscles of old HSP70 overexpressor mice compared with old WT mice may have occurred as a consequence of protection against age-related accumulation of products of oxidative damage and the preservation of the ability to activate redox-sensitive transcription factors such as NF-kB. Muscles of old WT mice demonstrated a chronic up-regulation of SOD and catalase activity. Despite this increased antioxidant defense enzyme activity, muscles of old WT mice also showed a fall in total glutathione content and increased MDA and protein carbonyl content, suggesting that oxidative stress remained elevated in these muscles. In addition, the ability of muscles of old WT mice to activate NF-kB-mediated transcription after a moderate and nondamaging exercise stress was severely blunted in comparison with muscles of adult WT mice. Evidence of ongoing oxidative damage may explain the inability of muscles of old WT mice to activate NF-kB-mediated transcription. Activation of NF-kB is dependant on a controlled increase in the production of reactive oxygen species (ROS). Lack of activation of NF-kB in muscles of old mice suggest that the signal for activation does not occur or that NF-kB may also be oxidatively damaged.
Lifelong overexpression of HSP70 provided protection against the age-associated accumulation of oxidative damage. Several studies have demonstrated increased HSP content of different tissues provides protection against ROS-mediated damage and that preservation of skeletal muscle during the aging process may occur via the same protective mechanism. The HSP70 content of skeletal muscle decreases with age and the ability of muscles to increase the production of HSPs after a variety of stresses, including oxidative stress, is also severely attenuated. Previous work from our laboratory has demonstrated that lifelong overexpression of HSP70 in skeletal muscle of mice significantly preserved muscle function, and data presented in the current study suggest that this preservation of muscle function occurs by preventing accumulation of oxidation products and preserving the ability of the muscle to activate ROS-mediated transcription after exercise stress. However, the causative link between HSP70 expression and protection against oxidative damage is yet to be fully elucidated.
The present findings suggest that the skeletal muscles from HSP70 overexpressor mice are protected from the accumulation of protein carbonyls with age. No age-related changes were observed in muscles from the HSP70 transgenic mice in any of the five proteins with molecular masses of 
75, 50, 35, 32, and 25 kDa, which showed increased levels of carbonyl content in muscles of old WT mice. Such alterations to cellular proteins may result in the loss of structural or enzymatic activity of the individual proteins, which will contribute to muscle dysfunction. In tissues of younger individuals, oxidized cellular proteins appear to be recognized and efficiently degraded by the proteasome and recent data demonstrate that proteosome activity is reduced in a variety of tissues, including skeletal muscle, of old animals. One possibility is that maintenance of HSP content of plays a major, though indirect, role in maintaining proteosome activity by reducing protein aggregation.
In summary, this study demonstrated that the dramatic improvement in the functional capacity of muscles of old HSP70 overexpressor mice compared with old WT mice may have occurred as a consequence of protection against age-related accumulation of products of oxidative damage and preservation of the ability of muscles to activate NF-kB–mediated transcription after stress. The fundamental mechanism by which HSP70 overexpression prevents accumulation of oxidation products is unclear, although further studies examining the role of HSPs in preservation of proteosome activity are warranted.
|
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4935fje
This article has been cited by other articles:
|
|
 |
|
  E. E. Dupont-Versteegden, R. Nagarajan, M. L. Beggs, E. D. Bearden, P. M. Simpson, and C. A. Peterson Identification of cold-shock protein RBM3 as a possible regulator of skeletal muscle size through expression profiling Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2008; 295(4): R1263 - R1273. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A. Gupte, G. L. Bomhoff, and P. C. Geiger Age-related differences in skeletal muscle insulin signaling: the role of stress kinases and heat shock proteins J Appl Physiol, September 1, 2008; 105(3): 839 - 848. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. V. Brooks, A. Vasilaki, L. M. Larkin, A. McArdle, and M. J. Jackson Repeated bouts of aerobic exercise lead to reductions in skeletal muscle free radical generation and nuclear factor {kappa}B activation J. Physiol., August 15, 2008; 586(16): 3979 - 3990. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. C. Kayani, G. L. Close, M. J. Jackson, and A. McArdle Prolonged treadmill training increases HSP70 in skeletal muscle but does not affect age-related functional deficits Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2008; 294(2): R568 - R576. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. Du, H. Li, Y. Bian, and Y. Zhong Heat-shock protein 90{alpha}1 is required for organized myofibril assembly in skeletal muscles of zebrafish embryos PNAS, January 15, 2008; 105(2): 554 - 559. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. R. Tupling, E. Bombardier, R. D. Stewart, C. Vigna, and A. E. Aqui Muscle fiber type-specific response of Hsp70 expression in human quadriceps following acute isometric exercise J Appl Physiol, December 1, 2007; 103(6): 2105 - 2111. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
